Our long term objective is to describe, and achieve a mechanistic understanding of, normal developmental and pathological retinal vasculogenesis. Hand in hand with this effort we will be developing rational approaches to the treatment and ultimate prevention of retinopathy of prematurity (ROP), a disease affecting a developing vasculature. As survival rates for premature infants has increased, ROP has become again a very significant cause of childhood blindness in developed countries. A causal association with exposure to a hyperoxic environment seems clear, but the mechanisms involved are not understood. Our studies are among the first to a elucidate a developmentally-regulated, testable, and biochemically-defined basis for the kinds of changes observed in human ROP infants. Our studies rely on the use of a well characterized canine model for the human disease. The neonatal canine retina is a perfect tissue to study normal developmental vasculogenesis, because it is only 60% vascularized at birth. When this developing system is exposed to hyperoxia for four days and then returned to normoxia, pathology similar to human ROP occurs. Like the human retinopathy, a major characteristic is florid neovascularization of the retina. The dog model of ROP is perhaps the best model of retinal angiogenesis. Our results so far have been enticing. We can clearly demonstrate the 2- and 3-dimensional characteristics of the retinal vasculature using our ATPase flat-embedding technique. We have measured gradients of the metabolite adenosine and source enzyme 5'nucleotidase (5'N) that correlate and exactly precede normal vascular development and pathological vasculogenesis in the disease state. Adenosine is a potent vasodilator, is chemotactic and mitogenic for endothelial cells, and is angiogenic on the chick chorioallantoic membrane. For these reasons we propose to investigate the role of adenosine in normal and pathological vasculogenic events and thus define the mechanisms behind the results obtained so far. The vasculogenic events will be analysed simultaneously for morphological changes and for functional changes and identification of cell types. The vascular cells involved will be identified using specific histochemical markers. Adenosine metabolism will be elucidated by 5'N histochemistry, adenosine immunohistochemistry, and A2 adenosine receptor binding. Histochemistry and receptor binding will be quantified morphometrically via image analysis. A potent inhibitor of 5'N will be used to determine the role of 5'N in both normal and abnormal retinal vasculogenesis. NECA, a potent adenosine agonist, will be used to determine the role of adenosine receptor mediated events in normal vasculogenesis. Finally, the effects of the adenosine angonists and antagonists will be tested directly on endothelial cells and pericytes in vitro. These studies will determine levels of correlation between the morphological events in retinal vasculogenesis and the metabolic changes to test whether or not adenosine is a regulator of these processes.